Selectivity, mainly governed by column chemistry, is the key in HPLC separation. Although reversed-phase columns (e.g., C18) are most commonly used in pharmaceutical applications, they often fail to retain highly polar molecules (e.g. counter ions), and offer limited selectivities. Ion exchange (IEX) chromatography is used to separate ionic or ionizable molecules. Ion pairing agents can be added to the mobile phase to tailor the separation capabilities for a variety of sample types, but this often results in extended equilibration time, a complicated mobile phase that is incompatible with MS, and a dedicated column.
Mixed-mode chromatography provides a viable solution to these challenges by using both reversed phase and ion-exchange retention mechanisms. One major advantage of this approach is that column selectivity can easily be modified by adjusting mobile phase ionic strength, pH and/or organic solvent concentration. As the result, not only is the selectivity of a mixed-mode column complementary to that of reversed-phase columns, but it also allows for the development of multiple complementary selectivities on a given column under different appropriate conditions. Mixed-mode chromatography is well-suited to retaining ionic analytes, hydrophobic (e.g. Naproxen) or hydrophilic (e.g. Na+ and Cl− ions), and requires no ion-pairing agents in the method, significantly improving MS compatibility. This technique has been growing rapidly because of its advantages over conventional chromatography, such as its high resolution, adjustable selectivity, high sample loading, and no need for ion-pairing agents. Many applications involving hydrophilic ionizable compounds that are problematic on a C18 column are easily addressed on a mixed-mode column.
Mixed-mode media can be classified into three categories based on column chemistry. The first type includes a blend of two different stationary phases (RP and IEX). The second type involves bonded silica, modified by a mixture of both RP and IEX ligands in the bonding step. Although these two materials seem straightforward to synthesize, their use in many applications is limited by selectivity drifting, mainly due to the difference in hydrolytic stability between the RP and IEX ligand bonded sites. Newer mixed-mode media (the third type) use functional silyl ligands that contain both RP and IEX functionalities to covalently modify silica particles. The constant ratio between RP and IEX bonded sites greatly improves the ruggedness of selectivity of the resulting phases so that more reliable chromatographic methods can be developed. This invention presents a new and single approach to make the third type of mixed-mode media using the epoxide ring-opening reaction.
Compositions useful in chromatographic separations, as well as methods of making and using these compositions, have been created and are described herein. Other embodiments, advantages and objects of the invention are set forth in the Detailed Description.